Association of Scrub Typhus in Children with Acute Encephalitis Syndrome and Meningoencephalitis, Southern India

Scrub typhus is an established cause of acute encephalitis syndrome (AES) in northern states of India. We systematically investigated 376 children with AES in southern India, using a stepwise diagnostic strategy for the causative agent of scrub typhus, Orientia tsutsugamushi, including IgM and PCR testing of blood and cerebrospinal fluid (CSF) to grade its association with AES. We diagnosed scrub typhus in 87 (23%) children; of those, association with AES was confirmed in 16 (18%) cases, probable in 55 (63%), and possible in 16 (18%). IgM detection in CSF had a sensitivity of 93% and specificity of 82% compared with PCR. Our findings suggest scrub typhus as an emerging common treatable cause of AES in children in southern India and highlight the importance of routine testing for scrub typhus in diagnostic algorithms. Our results also suggest the potential promise of IgM screening of CSF for diagnosis of AES resulting from scrub typhus.

Scrub typhus is an established cause of acute encephalitis syndrome (AES) in northern states of India. We systematically investigated 376 children with AES in southern India, using a stepwise diagnostic strategy for the causative agent of scrub typhus, Orientia tsutsugamushi, including IgM and PCR testing of blood and cerebrospinal fluid (CSF) to grade its association with AES. We diagnosed scrub typhus in 87 (23%) children; of those, association with AES was confirmed in 16 (18%) cases, probable in 55 (63%), and possible in 16 (18%). IgM detection in CSF had a sensitivity of 93% and specificity of 82% compared with PCR. Our findings suggest scrub typhus as an emerging common treatable cause of AES in children in southern India and highlight the importance of routine testing for scrub typhus in diagnostic algorithms. Our results also suggest the potential promise of IgM screening of CSF for diagnosis of AES resulting from scrub typhus.
central nervous system [CNS] by the pathogen) or encephalopathy without CNS invasion, such as in the case of severe systemic infection, metabolic derangement, or other neurologic complications after the infection (10,11). Identifying the pathogenesis could inform management and prognosis (10,12).
Early diagnosis is key to initiating prompt specific treatment, which can reduce complications and fatality rates of scrub typhus (2,13). Clinical diagnosis can be challenging because of the overlap of symptoms with other tropical infections endemic to the area that can also cause AES (5), such as dengue, chikungunya, malaria, and leptospirosis (14). Current microbiological diagnostics for scrub typhus, which are usually based on detecting IgM in serum samples or nucleic acid by PCR, have limitations. IgM appears in serum 5-6 days after onset of illness, can persist long after acute illness, and might cross-react with IgM of other cocirculating pathogens (14,15). Therefore, in AES patients with simultaneous microbiological evidence for another potential pathogen and O. tsutsugamushi, confirming O. tsutsugamushi as the cause is difficult. Detection of IgM in cerebrospinal fluid (CSF) is yet to be used widely in patients with suspected neurologic scrub typhus. Immunofluorescence assay has long been considered the reference standard serologic test, but its use is limited by expense and challenges in interpretation. PCR might help overcome shortcomings of serologic tests with respect to cross-reacting and persisting antibodies, but a positive result is only likely during the bacteremia phase of infection (16). Moreover, the recommended samples for O. tsutsugamushi PCR are blood or eschar material, whereas the sensitivity of PCR on CSF remains unclear (7,16,17). Therefore, a diagnostic approach using accessible tests to determine the association of scrub typhus with AES is urgently needed.
We present preliminary findings of an ongoing multicenter prospective cohort study suggesting scrub typhus as a cause of AES in children in southern India. We used a diagnostic strategy to investigate the association of scrub typhus with AES. We describe the clinical spectrum, epidemiology, and laboratory findings of children with scrub typhus manifesting as AES. We then identify patients demonstrating evidence of meningoencephalitis or encephalitis and explore the value of performing IgM ELISA on CSF samples.

Patients and Study Sites
We prospectively enrolled pediatric patients from 1 month to 18

Ethics Statement
The study was approved by the institutional ethics and review boards of the hospitals and the coordinating center, National Institute of Mental Health and Neurosciences. Full informed consent was taken by the study team, who were trained specifically in taking consent from caregivers, and assent from older children, using procedures and forms approved by the institutional ethics committees.

Clinical Assessment and Data Collection
Clinical coinvestigators (V.K.G., L.A.V., F.S.D., S.S., M.K.) from the 3 centers performed clinical and neurologic examination of patients. After obtaining consent, we entered detailed clinical history and examination findings on an electronic clinical proforma. Results of routine laboratory tests and patient demographics were collected and entered online by N.P., S.M., or T.D. We determined the normal range of routine laboratory tests according to the age of the patient (18) and defined single-organ dysfunction and multiorgan dysfunction syndrome according to established criteria (19).

Microbiological Testing
Blood and CSF specimens of enrolled patients were tested at the Department of Neurovirology, National Institute of Mental Health and Neurosciences, by using a laboratory algorithm designed by Ravi et al. (5) with some modifications ( Figure  1). First-line tests included serum IgM ELISA for various pathogens. CSF samples of patients with IgM-positive ELISA serum samples were diluted in 1:10 proportion for detection of IgM. We performed confirmatory tests on IgM-positive patients, including real-time PCR for O. tsutsugamushi on CSF and blood samples. For PCR, we extracted DNA from samples by using the QIAamp DNA mini kit (QIA-GEN, https://www.qiagen.com) and performed real-time PCR targeting the 47kDa protein gene using the protocol described by Jiang et al. (20). In addition, we also performed real-time PCR and IgM ELISA for O. tsutsugamushi on stored CSF samples of patients with a negative result after thirdline tests. We used the Scrub Typhus Detect IgM ELISA kit (InBios International, http://inbios.com) and considered an optical density (OD) cutoff of 0.8 in serum (15) and 0.5 in CSF (21) samples to be positive. Scrub typhus was diagnosed in patients with IgM-positive real-time PCR or ELISA.
The level of certainty of association of scrub typhus with AES in cases positive for >1 microbiological test(s) for O. tsutsugamushi was determined by using criteria determined by Granerod et al. (11) with modifications (Tables 1, 2). We identified patients with meningoencephalitis/encephalitis (ME) and scrub typhus ME as those demonstrating clinical signs of either encephalitis or meningoencephalitis ( Table 2).

Statistical Analysis
We performed statistical analysis by using R version 3.6.3 (The R Project for Statistical Computing, https:// www.r-project.org). We presented descriptive data for categorical variables as frequencies, percentages, or both and described continuous variables using mean +SD or median and interquartile range (IQR). To describe the diagnostic accuracy of CSF IgM, we compared results against CSF PCR to calculate the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of CSF IgM with 95% CI. We also calculated those values for patients with scrub typhus ME.
*AES, acute encephalitis syndrome; CSF, cerebrospinal fluid. †Without evidence of another potentially causative pathogen. ‡With evidence of another potentially causative pathogen.

Results
We included a total of 376 children with AES in the study (Appendix Figure 1). Of those, scrub typhus was diagnosed in 87 patients by using the laboratory algorithm described.

Microbiological Testing
We collected samples for microbiological testing a median of 11 (IQR 8-14) days from onset of symptoms and median of 4 (IQR 2-6) days after hospitalization.

ME and Scrub Typhus ME
Of the 87 patients, 65 (74.7%) had findings suggestive of ME (Appendix Tables 3, 4). The diagnostic association of ME with scrub typhus was confirmed or probable (single-positive) in 54 (62%) patients (Figure 2), and of those patients, 43 had ME. Therefore, among all 87 patients, 49.4% had scrub typhus ME (Figure 3).

Diagnostic Accuracy of CSF IgM Testing
We  (Figure 3).

Demographic and Clinical Profile
The male:female ratio of children with scrub typhus was 1.5:1. Ages ranged from 2 months to 17 years; the mean age was 8.5 (SD +4) years (Table 4). Proportions of AES-scrub typhus cases were highest in the months of August and September. In addition, the number of AES-scrub typhus patients and their proportion of total AES patients followed the same pattern as the total number of AES cases (Appendix Figures 2, 3). The largest percentage of children (37%) were from Anantapur district in Andhra Pradesh state, followed by 17% from Tumkur district in Karnataka state ( Figure  4). Nearly 48% of patients were referred from another hospital, and 34% received anti-infective medications before being admitted to the study hospital. The median duration of illness before admission to the study hospital was 6 (IQR 4-9.5) days. All 87 children experienced fever and change in mental state; fever was the first symptom in 95% of cases. Around 62% of children had seizures; generalized tonic-clonic seizures were the most common type (74%), and some patients also had focal, tonic, or absence seizures. Upon examination at the time of hospital admission, 55 (64%) patients had altered mental state. The Glasgow Coma Scale at admission ranged from 3 to 15; the median was 13 (IQR 10-15) ( Table 5). Signs of meningeal irritation were detected in 48% of patients, cerebellar signs in 21%, and papilledema in 20%. Other neurologic findings were cranial nerve abnormalities (6%), involuntary movements (9%) and photophobia (9%), abnormal tone (50%), decreased power (19%), and abnormal plantar reflexes (24%) ( Table 5). Approximately 39% of the patients met criteria for multiorgan dysfunction syndrome (Appendix Table 5).

Treatment
Of the patients with scrub typhus, 44 (51%) required care in the intensive care unit during their hospitalization, and 26 of those required ventilatory support. All patients except 1 were prescribed doxycycline (100 mg 2×/d for 10 days). One patient died during hospitalization.

Discussion
Our findings suggest that scrub typhus is a major cause of AES in children in southern India. Of 193 (51%) patients with a known etiology, a microbiological test for O. tsutsugamushi was positive in 87 (45%) 716 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 29, No. 4, April 2023w   (17,(25)(26)(27)(28). Although studies in India have documented meningoencephalitis as a manifestation of scrub typhus in children (2,29,30), our study highlights the importance of systematic screening for scrub typhus in children with AES in southern India. Scrub typhus is a well-recognized cause of acute febrile illness in the major southern Indian states of Andhra Pradesh and Karnataka (31-33), but we report scrub typhus is also a common cause of AES in children from these states. Given the challenges in clinical diagnosis (10,14,15) and complexity of defining the causal relationship of scrub typhus with AES on the basis of serum IgM ELISA, the most widely used test for scrub typhus (15), we used a causality strategy. This diagnostic strategy helped in differentiating the certainty of association of 87 AES-scrub typhus cases into 16 cases with confirmed association, 55 with probable association, and 16 with possible association. Real-time PCR, which is confirmatory for scrub typhus, was positive in 6/39 (15%) cases with microbiological evidence of another pathogen and increased the diagnostic association from possible to confirmed. We were able to diagnose scrub typhus in 1 extra case in which  IgM ELISA for O. tsutsugamushi and tests for other pathogens were negative. Despite systematic testing, the prevalence of positive real-time PCR in children with AES caused by scrub typhus was low in our study (16 [18%] children), although still higher than in other studies (7,34). PCR positivity might be maximized by collecting clinical samples sooner after illness onset and using whole blood or buffy coat instead of serum to capture intracellular bacteria (14,16). In this study, patients with a positive PCR had a median duration of illness of 9 (IQR 5.75-12.25) days before clinical specimen sampling versus 11 (IQR 8.5-14.5) days for patients with a negative PCR result. Because IgM does not ordinarily cross the blood-CSF barrier, presence of those antibodies in CSF implies their production within the CNS (35) and higher certainty of association with the infection compared to serum IgM. Using CSF IgM ELISA increased the certainty of association from possible to probable in 23 patients who had simultaneous evidence of another pathogen. Although the kit is recommended for *All 87 children had fever and change in mental status, but detailed clinical findings of just 86 children were recorded. †Change in mental status was defined as >1 of the following: change in cognition (such as confusion or disorientation), drowsiness, coma, lethargy, irritability, reduced activity, poor feeding, irrelevant/abnormal talk. ‡Includes cough and/or difficulty breathing. Both were present in 5 patients each. §Includes >1 of the following symptoms: abdominal pain, abdominal distension, and diarrhea. Individually, abdominal pain was a symptom in 19 (22%), abdominal distention in 12 (14%), and diarrhea in 3 (3.5%) patients. ¶Signs of respiratory distress occurred in 16 (19%) patients; reduced air entry or abnormal respiratory sounds occurred in 5 (5%) patients. #Hepatomegaly was present in 47 (54.7%) patients, splenomegaly was present in 16 (18.6%) patients, and ascites was present in 4 (4.7%) patients. **Four persons had abnormalities in 6th cranial nerve, and 1 in 7th cranial nerve. † †Opsoclonus and myoclonus, choreoathetoid movements and hemiballismus, abnormal perioral movements, lip smacking, teeth grinding, and rapid eye blinking occurred in 1 patient each; tremors occurred in 2 patients. ‡ ‡Includes >1 of the following: truncal ataxia, gait abnormality, finger-nose incoordination, nystagmus, dysdiadochokinesis, and dysarthria.
detecting IgM in serum samples only, Murhekar et al. (6) observed good correlation between OD values for O. tsutsugamushi IgM in serum and CSF. They determined a cutoff OD value of 0.22 after testing CSF samples from 374 children <14 years of age with AES in Gorakhpur, Uttar Pradesh state, India (35). A cutoff OD value for IgM in CSF has not been determined in the southern states in India, so we used a higher cutoff (0.5), as used by Behera et al. (21) for CSF of children with scrub typhus ME in eastern India.
Our results demonstrate that, compared with PCR, IgM ELISA of CSF had a sensitivity of 92.9%, but with a wide 95% CI, suggesting the estimate is less precise. Although the comparison is indirect, that sensitivity is similar to that of serum IgM by the same ELISA kit (92.4%) used for patients with acute febrile illness caused by scrub typhus in southern India (14). The specificity of CSF IgM ELISA was moderate compared to PCR at 82%. That finding might be because PCR positivity was less common in our study, which could be explained by delayed sampling during the course of illness, resulting in a higher likelihood of detection of IgM than DNA. In addition, the use of a single reference standard (PCR) in our study could result in a low PPV of IgM ELISA of CSF. The sensitivity of CSF IgM in patients with scrub typhus ME was 85.7%. Because only 11 patients did not have features suggestive of ME, ascertaining the true specificity is difficult.
Almost three quarters of the patients with AES-scrub typhus had meningoencephalitis. Distinguishing patients with scrub typhus ME from patients with encephalopathy with other causes is crucial. Therapeutic failure of doxycycline, the drug of choice for scrub typhus, has been reported in patients with scrub typhus ME (36). This failure could be caused by inadequate concentration of doxycycline in CSF at conventional doses and might indicate the need for increased dosages, intravenous administration, or administration of other antimicrobial agents such as rifampin that have good penetration to the CNS. However, the efficacy of this treatment is yet to be proven (37,38).
The neurologic manifestations in children with scrub typhus that meet the broader epidemiologic definition of AES are rarely reported (13,25,39,40), and no data from southern India have been published. Of all children with scrub typhus in our study, 8 (9%) had involuntary hyperkinetic movements that are rare neurologic manifestations of scrub typhus more often reported in adults than children (41). Opsoclonus-myoclonus, best recognized as part of opsoclonus-myoclonus-ataxia syndrome associated with neuroblastoma in children, is rarely caused by infections (13,41). Only 2 such cases of scrub typhus associated with pediatric opsoclonus-myoclonus-ataxia syndrome have been reported from India (42,43). Cerebellar signs, which are uncommon in children with scrub typhus (3,13), were noted in almost one fifth of the children in our study. As reported by Vishwanath et al. (30), the sixth cranial nerve was the most affected cranial nerve. Papilledema was detected in 20% of children in our study. Few studies have reported direct retinal involvement and isolated optic disc edema in the absence of raised intracranial pressure in scrub typhus (29,44,45); however, findings in this area remain inconclusive in our study. Presence of eschar typically occurs in 4%-46% of patients with scrub typhus; therefore, while specific, eschar is not a sensitive marker (30), and it was found in only 5% of patients in this study.
The first limitation of our study is that, whereas serum IgM ELISA is the most widely used specific test for O. tsutsugamushi, we used a single-positive IgM result as a criterion for diagnosis of scrub typhus. Obtaining serial blood samples and performing immunofluorescence or similar assays to demonstrate a 4-fold rise in antibody titers would have enabled more certainty in the diagnosis, especially in cases in which antibodies to another pathogen were detected. However, we defined those patients as having possible scrub typhus to allow for this uncertainty, and they comprised only 18% of the scrub typhus patients in this study. Also, for IgM detection in CSF, we relied on a cutoff value widely used for serum IgM, because a cutoff value for CSF has not been determined in this region. Furthermore, we could not perform sequencing of the PCR-amplified nucleic acid or characterization of surface antigen because of limited resources. In this study, despite limited accessibility and shortcomings of reference standard tests, we present a stepwise approach to identify scrub typhus as a probable or confirmed etiology by using tests that are relatively easy to access and perform. Our findings highlight the importance of systematic routine testing for the treatable and common pathogen O. tsutsugamushi in all patients with AES in southern India, as is practiced in several states in northern India. This testing could have a notable effect on the approach to clinical management and public health interventions for patients with AES. Apart from reinforcing common clinical, epidemiologic, and laboratory findings reported by other studies (13,29,39,40,46), we report insights into the neurologic spectrum of scrub typhus in children, which appears to be broad and underreported.
Finally, CSF IgM ELISA is a promising test for patients with AES caused by scrub typhus, which requires evaluation in a larger population and determination of a region-specific cutoff OD value. Combining CSF PCR with CSF IgM ELISA wherever feasible might increase the certainty of association between AES and scrub typhus.